The present invention relates to methods of treating viral infections, particularly HIV infection, using novel combinational therapy. The novel combinational therapy employs either the peptide DP-178, DP-107 or fragments, analogs and/or homologs thereof, and at least one other therapeutic agent.
DP-178 is a peptide corresponding to amino acids 638 to 673 of the HIV-1LA1 transmembrane protein (TM) gp41. DP-178 includes portions, analogs, and homologs of DP-178, all of which exhibit antiviral activity. Antiviral activity includes, but is not limited to, the inhibition of HIV transmission to uninfected CD-4+ cells. Further, the invention relates to the use of DP-178 and DP-178 fragments and/or analogs or homologs as inhibitors of retroviral transmission, in particular HIV, to uninfected cells, in both humans and non-humans. The present invention also relates to the antiviral peptide DP-107, a peptide corresponding to amino acids 558 to 595 of the HIV-1LA1 transmembrane protein (TM) gp4l, that are present in other enveloped viruses. More specifically, the invention is directed to the use of DP-107, fragments and/or analogs or homologs in combination with other therapeutic agents to treat viral infections, particularly HIV infection. Further, the invention encompasses novel pharmaceutical compositions comprising DP-178 or DP-107 and at least one other therapeutic agent.
The human immunodeficiency virus (HIV) is a pathogenic retrovirus and the causative agent of acquired immune deficiency syndrome (AIDS) and related disorders (Barre-Sinossi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinossi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 223:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4+ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and untimely death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984; RNA Tumor Viruses, Weiss, R. et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1,-II,-III), and feline leukemiavirus. The HIV viral particle consists of a viral core, made up of proteins designated p24 and p18. The viral core contains the viral RNA genome and those enzymes required for replicative events. Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kD precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains noncovalently associated with gp41, possibly in a trimeric or multimeric form (Hammerwskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD-4+ T lymphocytes because the CD-4 surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312: 767-768, Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD-4+receptor molecules, while gp41 anchors the envelope glycoprotein complex in the viral membrane (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348) and thus explains HIV""s tropism for CD-4+ cells.
HIV infection is pandemic and HIV associated diseases represent a major world health problem. Although considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the viral life cycle have been considered targets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). Intervention could potentially inhibit the binding of HIV to cell membranes, the reverse transcription of HIV RNA genome into DNA or the exit of the virus from the host cell and infection of new cellular targets.
Attempts are being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has been on CD-4+, the cell surface receptor for HIV. For example, recombinant soluble CD-4 has been shown to block HIV infectivity by binding to viral particles before they encounter CD-4 molecules embedded in cell membranes (Smith, D. H. et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates are relatively less sensitive to inhibition by recombinant CD-4 (Daar, E. et al., 1990, Ann. Int. Med. 112:247-253). In addition, recombinant soluble CD-4 clinical trials have produced inconclusive results (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989, Proc. Vth Int. Conf. on AIDS, p564, MCP 137).
The virally encoded reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T, have been developed which have also been shown to be active against HIV (Mitsuya, H. et al., 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
The late stages of HIV replication, which involve crucial virus-specific secondary processing of certain viral proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erikson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, these proteins seem to be the most promising candidates to act as antigens for anti-HIV development. To this end, several groups have begun to use various portions of gp16O, gp120, and/or gp41 as immunogenic targets for the host immune systems. See for example, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al., WO 92/22, 654; Schafferman, A., WO 91/09,872; Formoso, C. et al., WO 90/07,119. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Recently, double stranded RNAs, which elicit a general immune response; have been used in combination with antivirals such as interferon, AZT and phosphonoformate to treat viral infections. See Carter, W., U.S. Pat. No. 4,950,652. In addition, a therapy combining a pyrimidine nucleoside analog and a uridine phosphorylase inhibitor has been developed for the treatment of HIV, see Sommadossi, J. P. et al., U.S. Pat. No. 5,077,280. Although these specific therapies may prove to be beneficial, combination therapy in general has the potential for antagonism as demonstrated in vitro with azidothymidine (AZT) and ribavirin. See U.S. Pat. No. 4,950,652. Moreover, combination therapy is potentially problematic given the high toxicity of most anti-HIV therapeutics and their low level of effectiveness. Thus, there is a need for a combination therapy which is effective yet non-toxic.
The present invention provides a novel combination therapy based on the use of viral fusion inhibitors (DP-178 and DP-107, etc.) in combination with other antivirals. DP-178 and DP-107 are both novel therapeutics in that they prevent the virus from fusing with the cell, thereby very effectively preventing cell to cell transmission of the virus. In addition, DP-178 and DP-107 have proven to be non-toxic in in vitro studies and in animals. The present invention provides the first reported use of such peptides in combination with another antiviral or any other therapeutic agent.
The present invention relates to methods of treating or preventing viral infections, in particular HIV infections, in mammals, including humans, by administering an effective amount of DP-178, or a pharmaceutically acceptable derivative thereof in combination with at least one other therapeutic agent.
The present invention also relates to methods of treating or preventing viral infections, in particular HIV infections, in mammals, including humans, by administering an effective amount of DP-107 or pharmaceutically acceptable derivatives thereof in combination with at least one other therapeutic agent.
More specifically, the invention relates to methods of treating or preventing viral infections in mammals, including humans, by administering an effective amount of DP-107, DP-178, or a pharmaceutically acceptable derivative thereof, in combination with at least one other antiviral agent. The invention includes the administration of the active agents, e.g., DP-107, DP-178 or another antiviral either concomitantly or sequentially, including cycling therapy. Cycling therapy involves the administration of a first antiviral compound for a period of time, followed by the administration of a second antiviral compound for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies. The invention encompasses combinations of DP-107, DP-178 or a pharmaceutically acceptable derivative thereof and at least one other therapeutic, particularly another antiviral, that are synergistic, i.e., better than either agent or therapy alone.
The invention also encompasses combinations of DP-178, DP-107 or a pharmaceutically acceptable derivative thereof with a least one other antiviral having a different site of action than the viral fusion inhibitor. Such a combination provides an improved therapy based on the dual action of these therapeutics whether the combination is synergistic or additive.
The present invention is also directed to methods of treating or preventing HIV infection in mammals, including humans, by administering an effective amount of DP-107, DP-178 or a pharmaceutically acceptable derivative thereof in combination with at least one other therapeutic agent, in particular at least one other antiviral.
The novel antiviral combinations of the present invention provide a means of treatment which may not only reduce the effective dose of either drug required for antiviral activity, thereby reducing toxicity, but may also improve the absolute antiviral effect as a result of attacking the virus through multiple mechanisms. Similarly, the novel antiviral combinations provide a means for circumventing the development of viral resistance to a single therapy, thereby providing the clinician with a more efficacious treatment.
Another aspect of the invention encompasses pharmaceutical compositions and formulations for treating or preventing viral infections, in particular HIV infections, wherein said compositions comprise an effective amount of DP-178, DP-107, or a pharmaceutically acceptable derivative thereof, at least one additional therapeutic agent and a pharmaceutically acceptable carrier.
Therapeutic agents to be used in combination with DP-178, DP-107 or a pharmaceutically acceptable derivative thereof encompass a wide variety of known treatments. Preferably, the combinations employ DP-107 or DP-178 in combination with agents with a different mode of attack. Such agents include but are not limited to: antivirals, such as cytokines, e.g., rIFN xcex1, rIFN xcex2, rIFN xcex3; inhibitors of reverse transcriptase, e.g., AZT, 3TC, D4T, ddI, and other dideoxynucleosides or dideoxyfluoronucleosides; inhibitors of viral mRNA capping, such as ribavirin; inhibitors of HIV protease, such as ABT-538 and MK-639; amphotericin B as a lipid-binding molecule with anti-HIV activity; and castanospermine as an inhibitor of glycoprotein processing.
Thus, the present invention provides an improved antiviral therapy for treating a broad spectrum of viruses including HIV.
The present invention also provides combinational therapy which yields improved efficacy over either agent used as a single-agent therapy.
In addition, the invention provides combinational therapy which allows for reduced toxicity of DP-178 and DP-107 and/or the therapeutic agent with which the peptides are used; thereby providing a higher therapeutic index.
The instant invention provides a combinational therapy which provides a means for circumventing the development of viral resistance to a single therapy.
As used herein, the term xe2x80x9cviral infectionxe2x80x9d describes a diseased state in which a virus invades healthy cells, uses the cell""s reproductive machinery to multiply or replicate and ultimately lyse the cell resulting in cell death, release of viral particles and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible result of viral infection.
As used herein, the term xe2x80x9ctreating or preventing viral infectionsxe2x80x9d means to inhibit the replication of the particular virus, to inhibit viral transmission, or to prevent the virus from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by the viral infection. The treatment is considered therapeutic if there is a reduction in viral load, decrease in mortality and/or morbidity.
The term xe2x80x9csynergisticxe2x80x9d as used herein refers to a combination which is more effective than the additive effects of any two or more single agents. A synergistic effect as used herein refers to the ability to use lower amounts (doses) of either single therapy to treat or prevent viral infection. The lower doses result in lower toxicity without reduced efficacy. In addition, a synergistic effect can result in improved efficacy, i.e., improved antiviral activity. Finally, synergy may result in an improved avoidance or reduction of viral resistance against any single therapy. A determination of a synergistic interaction between DP-178 or DP-107, and another therapeutic agent may be based on the results obtained from the antiviral assays described in Section 5.5. The results of these assays are analyzed using Chou and Talalay""s combination method (Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55) and xe2x80x98Dose-Effect Analysis with Microcomputersxe2x80x99 software (Chou and Chou, 1987, software and manual. p19-64. Elsevier Biosoft, Cambridge, UK) in order to obtain a Combination Index. Combination Index values  less than 1 indicates synergy, values greater than 1 indicate antagonism and values equal to 1 indicate additive effects.
The results of these assays are also analyzed using the method of Pritchard and Shipman (Pritchard and Shipman, 1990, Antiviral Research 14: 181-206). This computer program through three dimensional graphic analysis of the results allows for a determination of a synergistic or antagonistic interaction between the antiviral agents.
The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert and is not toxic to the patient to whom it is administered.
As used herein the term xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d refers to any homolog, analog, or fragment corresponding to the DP-178 or DP-107 peptides as described in Section 5.1.2. infra which exhibits antiviral activity and is relatively non-toxic to the subject.
The term xe2x80x9ctherapeutic agentxe2x80x9d refers to any molecule, compound or treatment, preferably an antiviral, that assists in the treatment of a viral infection or the diseases caused thereby.
Peptides are defined herein as organic compounds comprising two or more amino acids covalently joined by peptide bonds. Peptides may be referred to with respect to the number of constituent amino acids, i e., a dipeptide contains two amino acid residues, a tripeptide contains three, etc. Peptides containing ten or fewer amino acids may be referred to as oligopeptides, while those with more than ten amino acid residues are polypeptides.
Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
A (alanine)
R (arginine)
N (asparagine)
D (aspartic acid)
C (cysteine)
Q (glutamine)
E (glutamic acid)
G (glycine)
H (histidine)
I (isoleucine)
L (leucine)
K (lysine)
M (methionine)
F (phenylalanine)
P (proline)
S (serine)
T (threonine)
W (tryptophan)
Y (tyrosine)
V (valine)